laccore sop series biogenic silica analysis purpose...

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LacCore SOP series

Biogenic Silica Analysis

Purpose

To determine the percent biogenic silica in sediments using sodium hydroxide dissolution of silica and molybdate blue

spectrophotometry.

Principle

A freeze dried sediment sample is placed in hot sodium hydroxide to dissolve the silica in the sample. The dissolved

silica comes from biogenic sources, i.e. diatoms, and non-biogenic sources, i.e. clays. Timed aliquots are extracted from

the sodium hydroxide and dyed molybdate blue. Spectrophotometry is used to determine the samples absorption at 812

nm. The absorption percent is then used to calculate the percent biogenic silica in the sample.

Safety Precautions

When preparing reagents, take care to avoid spills, skin contact, eye contact, inhalation, or ingestion.

You will be working with strong acids and bases. Use protective gear, such as safety goggles, vinyl gloves, lab coat, etc.

When handling strong acids you must work in the fume hood.

During sample digestions, the water bath will be hot and will contain hot liquid. The digestion tubes will contain hot NaOH.

Take appropriate safety measures.

Label and date everything! Do not pour reagents from their original bottles into unlabeled or mislabeled bottles. Reagents

prepared by you must be stored in properly labeled containers. Label water bottles appropriately.

Notes

When using the microliter pipette, it is critical that the amount of sample you draw into the pipette and the amount expelled

from the pipette is always exactly the same. Pay attention to the level of liquid in the tip, and start over if the amount of

liquid doesn’t look quite right or if bubbles are present. Problems commonly arise from improperly seating the tip on the

pipette and from failing to depress the plunger completely.

When using the dispensette, it is also critical that the amount of sample you draw into the dispenser and the amount

expelled from the dispenser is always exactly the same. Just as the same person should do all of the pipetting, the same

person should do all of the dispensing with the dispensette so the all of the sample are treated the same. When using the

dispenser be sure to work at a moderate pace and always lift up on the plunger and depress the plunger in the same way.

Be sure to fully depress and lift up on the plunger to completely fill the chamber, taking care to be gentle. The first two to

three pumps need to be dispensed into the hazardous waste, to ensure that the plunger is completely full and all air is

removed from the lines. You can look through the plunger chamber and see if it has any bubbles, don’t dispense any

chemicals until all the air is gone. Between batches the dispensettes should not be used for storing chemicals for two

reasons, (1) it is not clear how or if evaporation of the sodium hydroxide will affect results and (2) glass is a component of

the dispenser. While exposing the glass to sodium hydroxide for a short time (less than 30 seconds) during dispensing

has not been shown to change results, sodium hydroxide being stored in a dispenser may lead to dissolution of the glass.

When done using a dispenser, always fill the container attached to the dispenser with high purity water to rinse it out and

dispense the water a few times to clean out the plunger.

The same technician should work on all the samples submitted by a researcher from the start of this procedure to the end.

The one exception is that standards made by one technician can be shared with another technician as long as the same

set of standards is used for all of the batches submitted by a researcher.

Use polyethelene products unless otherwise noted. Use of glass may contaminate the samples.

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Have all materials required pre-labeled and set up prior to beginning the digestions.

Use silica free water for all mixtures (Water from a high purity deionized water system, such as Milli Q water).

Always add the sample to the molybdate (never the reverse). This will help to keep the mixture above a certain pH.

If your particular spectrophotometer cannot be set at 812 (near infrared), a wavelength of 725 nanometers has been

suggested. As a result, a small reduction in sensitivity should be expected.

Sample preparation

1. Sample cores at a volume that will produce approximately 0.03g of DRY sediment. Generally, this will be enough

sample for an analysis. Samples with very low (< ~4%) biogenic silica will require additional dry sediment; those

with very high (> ~25%) biogenic silica may require less dry sediment. Additional sample is required for

duplicates, 17% duplicate analysis is typical.

2. Freeze the sediment samples, and place them into the lyophilizer (freeze-dryer) for at least 24 hours, until

completely dry.

3. Grind the samples to a fine powder with a mortar & pestle.

4. Place in 1 dram vials with caps for storage.

Standard Preparation

1 yellow Polycon 1 polyethylene volumetric flask, 1 L

Sodium Fluorosilicate, 1 g high purity deionized water

1 Polyethylene bottle, 1 L 9 polyethylene bottles, 50 mL

Standards are run each time samples are analyzed in order to set up a calibration curve. You must use high purity

deionized water. Polyethylene bottles are recommended, as glass will contaminate the standards.

1. Place approximately 1g of sodium fluorosilicate (Na2SiF6) in an open polycon and place the polycon in a

desiccator overnight to remove excess water. Do not heat or fuse.

2. Fill a polyethylene 1L volumetric flask two thirds full with high purity deionized water. Precisely weigh out 0.5642 g

of the sodium fluorosilicate and then add it to the flask. Be sure to rinse the weighing container into the flask.

3. Swirl occasionally to dissolve. Complete dissolution will take 30 minutes, do not rush.

4. After a minimum of 30 minutes (check to be sure that the sodium fluorosilicate has dissolved) fill the volumetric

flask to the fill line. The concentration of this standard is 3000 μmoles/L.

5. Using a 50 mL volumetric flask, begin with the following amounts of primary standard. Bring each to 50 mL total

volume. Standards are stored in the fridge when not in use. Be sure to start with a clean and dry volumetric flask

each time you make a standard. Unused sodium fluorosilicate solution can be put down the drain; it is what some

cities use to fluorinate the drinking water.

Primary standard

(mL)

Concentration

(μmoles/L)

.5 30

1 60

2 120

4 240

6 360

8 480

10 600

15 900

20 1200

6. Perform the analysis procedure as described in the Analysis Procedure section below.

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The analysis procedure should be repeated 2-3 times to start to establish average values for each standard. It is

important to keep track of the absorption values for each concentration, every time a batch of samples is run. A

list should be maintained in the spreadsheet. Rows 18-39 are for batch 1-11 results, these results should be

recorded in rows 23 and up. The spreadsheet maintains the statics for each batch of samples and the standard

deviations should be used as a guide when deciding if a point should be thrown out.

Fig 1: Spreadsheet for recording absorbance of the standards.

Record the results in the standard spreadsheet. You may wish to graph the results as well.

Preparing Reagents All work should be done under the fume hood. Be sure to label everything with name and date. Reagents Required: Ammonium Paramolybdate [(NH4)6Mo7O24 · 4H2O] Oxalic Acid Dihydrate [(COOH)2 · 2H2O] Anhydrous Sodium Sulfite [Na2SO3] Hydrochloric Acid (12N, 37%) [HCL] Metol (p-methylaminophenol sulfate) [C14H20N2O6S] Sodium Hydroxide [NaOH] Sulfuric Acid [H2SO4]

Sodium Hydroxide Solution: Needed for Day 1

1000 mL polyethelene volumetric flask High purity deionized water

20 g 0.5M Sodium Hydroxide

1. Add approximately 750 mL of high purity deionized water to a 1000 mL volumetric flask.

2. Add and dissolve 20 g sodium hydroxide (NaOH).

3. Add enough high purity deionized water to bring to a final volume of 1000 mL.

4. Empty flask into the 0.5 M NaOH dispensette.

5. This makes enough for 24 samples, repeat entire process for 44 sample batch size.

6. For large sample requests make up enough solution for the whole project at once.

7. Using the Dispensette, dispense 38.0 mL NaOH solution into a 50 mL digestion tube. Cap the digestion tube and

place in a centrifuge rack. Each tube cap should be assigned and labeled with a number (1 to 44, if doing multiple

batches start back over at 1 for each batch). Step 7 does not need to be done ahead of time. It may be done on day 2

while waiting for the hot water bath to heat up.

Molybdate Solution


4 g Ammonium paramolybdate 12 mL 12N Hydrochloric acid (37%)

1. Add approximately 300 mL of high purity deionized water to a 500 mL polyethelene volumetric flask.

2. Add and dissolve 4 g ammonium paramolybdate.

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3. Add 12 mL 12N HCL with a Repeater pipette, and swirl to mix.

4. Add enough high purity deionized water to bring to a final volume of 500mL.

5. Store in a dark plastic bottle; this solution is stable for approximately 4 months. Discard if greenish crystals precipitate

onto container walls (dissolve crystals with dilute sodium hydroxide).

Metol-Sulfite Solution (must be done at least one day before needed)


6 g Anhydrous sodium sulfite 10 g Metol (p-methylaminophenol Sulfate)

No. 1 Watman filter paper

1. Add approximately 300 mL high purity deionized water to a 500 mL polyethelene volumetric flask.

2. Add and dissolve 6 g Anhydrous Sodium Sulfite.

3. Add and dissolve 10 g Metol (p-methylaminophenol Sulfate [also known as paramethylaminophenol sulfate or 4-

methylaminophenol sulfate]).

4. Add enough high purity deionized water to bring to a final volume of 500 mL.

5. Filter solution through a No. 1 Watman filter paper and store in a clean, dark glass bottle, wrapped in aluminum foil.

6. This solution deteriorates rapidly and should be prepared fresh at least every month. Discard if brownish precipitates

form on container (dissolve with dilute sodium hydroxide).

Note: Metol goes by many names, some of which are: N-methyl-p-aminophenol sulfate, p-(methylamino)phenol sulfate,

monomethyl-p-aminophenol hemisulfate.

Oxalic Acid Solution (must be done at least one day before needed)

1000 mL polyethelene volumetric flask 500 mL High purity deionized water

50 g analytical grade reagent oxalic acid dihydrate

1. Measure 500 mL high purity deionized water and pour it into a 1000 mL polyethelene volumetric flask.

2. Add and dissolve 50 g analytical grade reagent oxalic acid dihydrate (it is okay if some of the crystals spill or don’t get

into the flask – there will be excess). Shake for 5-10 minutes. Let stand overnight. Do not fill the flask to 1000 mL.

3. This solution is supersaturated so you need to decant the solution from the crystals into a glass container. If all the

crystals dissolve within 24 hours, throw out the solution and remake.

4. This solution is stable indefinitely.

Reducing Solution (must be made the day of use)

Ratio: 5 mL Metol sulfate : 3 mL Oxalic acid : 3 mL Sulfuric acid : 4 mL high purity deionized water

For a batch size of 4 samples (7 extractions per sample + 10 standards + 1 blank)

250 mL wide-mouth polyethelene reagent polyethylene bottle 50 mL Metol sulfate solution

30 mL Oxalic acid solution 30 mL 50% Sulfuric acid solution

40 mL High purity deionized water (make to a volume of 150 mL)

For 12 samples (7 extractions per sample + 10 standards + 1 blank)

500 mL polyethelene volumetric flask 100 mL Metol sulfate solution


80 mL High purity deionized water (make to a volume of 300 mL)

For 24 samples (7 extractions per sample + 10 standards + 1 blank)

1 Liter polyethelene volumetric flask 200 mL Metol sulfate solution


160 mL high purity deionized water (make to a volume of 600 mL)

For 44 samples (4 extractions per sample + 20 standards + 2 blanks)

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1L polyethelene volumetric flask

208.4 mL Metol Sulfate solution 125.1 mL Oxalic acid solution

125.1 mL Sulfuric acid solution 166.7 mL High purity deionized water

For 88 samples (4 extractions per sample + 40 standards + 4 blanks)

1L polyethelene volumetric flask

416.8 mL Metol sulfate solution 250.2 mL Oxalic acid solution

250.2 mL Sulfuric acid solution 333.4 mL High purity deionized water

Use polyethelene volumetric flasks and the Eppendorf Repeater pipette (with a separate labeled combitip for each

reagent) to accurately measure the reagents. Each reagent should have its own labeled volumetric flask for this step.

These flasks are different from the volumetric flasks used to make the individual reagents.

Fig. 2: Supplies needed to make up reducing solution.

To accurately measure the reagents: Pour from the storage container or smaller vessel into a volumetric flask to the

nearest 50 or 100 mL. Use a plastic pipette to add the necessary amount of reagent so that the bottom of the meniscus is

on the line on the flask. Empty the flask into the brown reducing solution container. Add remaining (amount not in 50 or

100 mL intervals) reagent with the repeater pipette to the nearest half mL.

Figs 3-5: (3) Using the Pipette to accuratly fill the volumetric flask, (4) transfer the reagent to the brown reducing

soltion storage container and (5) use the repeater pipette to measure out the remaining reagnets.

1. Measure Metol solution and add to the brown 1 L bottle.

2. Add oxalic acid and mix.

3. Add slowly, while mixing, the sulfuric acid.

4. Add high purity deionized water and mix. (Be aware that the bottle will warm up during this procedure!)

5. Place reducing solution into the designated dispensette with the brown opaque nalgene bottle.

Sample Digestions

Sodium hydroxide filled digestion tubes Samples

Hot water bath Microliter pipette with Epitips

NOTE: Timing is very important. When loading samples or extracting aliquots you need to spend the same amount of

time on each sample. Spend 30 seconds per sample, so that the whole loading/aliquot procedure for all 44 samples takes

22 minutes. This is important because each individual sample must be removed 60, 90,120 and 200 minutes after it was

loaded. Use a timer to keep track of elapsed time. Watch the seconds and always remove a digestion tube from the hot

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water bath at 0 seconds and 30 seconds. If you get behind when loading a sample or removing an aliquot, skip that

sample so that the other aliquots are still removed on time. Record skipped samples if possible.

NOTE: When using the microliter pipette, it is critical that the amount of sample you draw into the pipette and the amount

expelled from the pipette is always exactly the same. Pay attention to the level of liquid in the tip, and start over if the

amount of liquid doesn’t look quite right or if bubbles are present. Problems commonly arise from improperly seating the

tip on the pipette and from failing to depress the plunger completely.

1. Turn on hot water bath to 85˚ C. 2. Place the racks of NaOH into the hot water bath. See the section on making reagents if the racks are not already set

up.

Fig. 6: NaOH racks set up, ready for the hot water bath.

3. Weigh the appropriate amount (typically 0.03 ± 0.005 g) of sample into a 1-dram shell vial cap. Do not touch the cap

with your fingers, use tweezers so that the oil from your fingers does not change the weight of the cap. Record core,

depth, weight, and sample number (1-44) in the biogenic silica spreadsheet. Weigh 44 samples per batch, making

sure to add duplicates as shown in the spreadsheet. It is handy to mark the cap of the vial with a dot for each time you

take a sample from it so that you know which samples have been duplicated. This step should not be done more than

a day or two in advance. When done in advance, weighed samples must be kept in a desiccator.

Fig. 7: Freeze dried sediment weighed out into vial caps and placed in numbered tray.

Ready to be added to the NaOH digestion tubes.

4. Set up the extraction bottles in the trays. You will need four bottles for each sample, one for each aliquot extraction

plus two bottles for blanks. Extractions are to be taken at 60, 90, 120, and 200 minutes. The trays should be labeled

for each sample number and extraction time. This avoids the need to label bottles.

Fig. 8: Extraction bottles set up in labeled trays.

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5. Once the hot water bath is up to temperature, place the first vial cap of weighed sediment into the appropriate NaOH

digestion tube, cap, and shake the tube. Don’t just pour the contents of the cap into the digestion tube but add the

whole cap and sample to the digestion tube. You have 30 seconds.

Fig. 9: Drop the sample and vial cap directly into the NaOH digestion tube.

6. Repeat step five for sample #2, #3, etc. Remember, you need to spend the same amount of time loading and shaking

each sample in 30 seconds intervals.

7. When loading is complete, return to the first sample and shake the sample to homogenize. Repeat for all samples in

order.

8. Using the Dispensette, measure 4 mL of high purity deionized water into each extraction bottle. You should only add

the water to the extraction bottles that you will be using in the next time interval so that evaporation or dust in the air

does not change your results. On the side set up 2-3 extra vials with high purity deionized water, in case of any errors

these vials will be ready for use.

9. After exactly 60 minutes, use the Eppendorf Reference microliter pipette (set to 1054) to extract a 1 mL aliquot from

each digestion tube and add it to the corresponding bottle of 4 mL high purity deionized water. Loosely cap the

extraction vial.

Fig. 10: Use a microliter pipete to remove the timed aliquots from the digestion tube.

10. After extracting all of the aliquots in each time interval briefly remove each digestion tube from the hot water bath,

shake, and return to the hot water bath.

11. After all extractions are finished in one time interval, tighten the caps on the extraction bottles.

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12. Repeat step 8, then steps 9 through 11 at exactly 90, 120 and 200 minutes. Additionally, shake each digestion tube at

150 and 180 minutes (between the extractions taken at 120 and 200 minutes).

13. When all the aliquots have been removed at T200 remove one blank aliquot, from each rack, in the same manner as

the sample aliquots. Record the times in your lab notebook using the sample digestion time chart below.

Sample Digestion Timing (Tminutes)

Add sample T0:___________

Remove 1st aliquot T60:__________

Remove 2nd

aliquot T90:__________

Remove 3rd

aliquot T120:_________

Shake digestion tubes T150:_________

Shake digestion tubes T180:_________

Remove 4th aliquot T200:_________

Analysis Procedures

Repeater pipette 196 plastic vials and caps

high purity deionized water Molybdate solution

Reducing solution *must be prepared fresh on the day of analysis*

NOTE: When using the microliter pipette, it is critical that the amount of sample you draw into the pipette and the amount

expelled from the pipette is always exactly the same. Pay attention to the level of liquid in the tip, and start over if the

amount of liquid doesn’t look quite right or if bubbles are present. Problems commonly arise from improperly seating the

tip on the pipetter and from failing to depress the plunger completely.

1. Turn on the spectrophotometer to warm up. The switch is located on the back, by the power cord.

2. Set up a parallel set of plastic vials and caps in the fume hood: one for each of your bottles from day 1 and one for

each primary standard concentration and one for the blank.

3. Pipette 2.0 mLs of Molybdate solution, using an Eppendorf Repeater pipette, into each of the empty plastic vials

(begin with the blank, then the standards from lowest to highest concentration, then the samples in order).

4. Pipette 0.5 mL of sample into respective vials with Eppendorf microliter pipette (set the pipette to 0502). Record the

time after pipetting the last sample. The used pipette tips can be tossed in the trash. Remember to pipette another

round of standards after sample 22 (beginning of tray 3).

5. Dispense 4.0 mL of silica free distilled water (high purity deionized water) into each vial during the 15-minute waiting

period begun upon pipetting the last of the samples in step 4 (Use the dispensette).

Fig. 11: Use the dispensette to add water and reducing solution to the samples.

The dispensette has a long hose to reach all of the trays.

6. After 15 minutes (time required for faint yellow color to develop, but no more than 30 minutes), add 3.0 mL of reducing

solution to the vials in the same order in which the sampling was done (use the dispensette). NOTE THE TIME. If

time exceeds 30 minutes you must start over because, “undesirable changes in the isomeric form of the

silicomolybdate complex will take place,” according to Strickland and Parsons (1972).

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Fig. 12: Faint yellow color of the sample that developes before the addition of the Reducing solution.

7. Cap vials to reduce evaporation, shake lightly, and wait at least 3 hours (but no more than 5 hours) for blue color to

develop. Samples must be run in the spec within 6 hours, if you wait more than 5 hours before starting to run samples

in the spec you may not get all the samples run before 6 hours is up. After adding the reducing solution to all

samples, clean the Dispensettes by drawing several rounds of high purity DI water through them.

Sample Digestion Timing

Added last sample to Molybdate T0:___________

Add reducing solution T15:__________

Run in spectrophotometer T180:__________

8. On the spectrophotometer, run the sipper tubing back through the SuperSipper.

9. In the menu, move down to the “Library,” press enter.

Fig. 13: Spectrophotometer main menu.

10. Move down to BSIII, press enter.

Fig. 14: Library menu.

11. Press run twice.

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Fig. 14: Run the BSIII program. Fig. 15: Main page of the BSII program.

12. Once you are in the BSIII program you can run the program by activating the sipper with a light tap on the silver plate.

Fig. 16: BSIII program ready for use.

13. Draw several rounds of high purity deionized water through the sipper/spec. Use the blank sample to zero the

instrument before analyzing your samples.

Fig. 17: SuperSipper attachment on the spectrophotometer.

14. Analyze the samples in the same order: standards from lowest to highest concentration, then samples in order. Finish

the first two trays of samples before beginning the third and fourth trays of samples.

Fig. 18: Set up of samples, sipper/spec and hazardous waste collection jug. It is helpful to prop the samples up on a

box or block of wood to prevent kinking the tubing on the spectrophotometer.

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15. Record the absorbance values for each sample. Record the results in the spreadsheet, using whole numbers to enter

the absorbance in the orange boxes. For example, if the spec says 0.045 enter 45 into the spreadsheet. This is

designed to save time by not having to add extra zeros and decimal points. See section below about using the

spreadsheet.

16. When each sample is finished, empty the vial into the hazardous waste container. Stack the vials in the large wash

bin so they are ready to be washed. The vials need to be stacked in an orderly fashion to get cleaned properly; the

opening of one vial should point towards the bottom of an adjacent vial.

17. After all samples are finished, draw several rounds of high purity deionized water though the sipper/spec. Disconnect

the tubing from the super sipper.

18. Waste must be labeled and taken to the Pollen lab for removal by DEHS.

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Using Excel

List of spreadsheets:

ARF Analysis Request Form, list of samples

Batches List of batches, breakdown of how the samples form batches

Stds List of standards, standard statistics

B1 Batch 1 data

B2 Batch 2 data

B3 Batch 3 data

B4 Batch 4 data

B5 Batch 5 data

B6 Batch 6 data

B7 Batch 7 data

B8 Batch 8 data

B9 Batch 9 data

B10 Rep. Batch 10 repeats only data

B11 Rep. Batch 11 repeats only data

Cum. Res. Cumulative results from all batches

Cum. Res. Chart Cumulative results in chart form

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ARF: Analysis Request Form, list of samples

Copy the first three columns of the ARF and paste (paste special: values) into the first three columns on this page. Type in

the name of the person that the samples are for. The name carries over to the standards spreadsheet for record keeping.

Good for up to 337 samples (9 batches). If you get a message about cells being locked you have exceeded the 337

sample limit. Copy and paste only 337 samples into this spreadsheet. Start a new workbook for additional samples.

Only the orange cells on this spreadsheet can be edited.

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Batches: List of batches, breakdown of how the samples form batches

The samples are automatically placed in batches with repeats. This spreadsheet is auto fill only and cannot be edited. It

shows the breakdown of how each batch is determined. For the cumulative results spreadsheet to work the batches

cannot be changed.

Bold numbers are repeats. The last four of each batch is repeated as the first four in the next batch. The last sample of

tray 2 is same as the first sample of tray 3 (because the standards change). Plus two additional repeats per set of

standards.

There are 337 samples in 9 batches, with 59 of them being repeats. Approximately 17% repeats, not including any

additional repeats from samples that did not turn out (can be manually entered in batches 10 and 11, and only batches 10

and 11). Batches 10 and 11 are designed to be all manual enter. Batches 10 and 11 can be used without all of the

previous batches being used.

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Stds: List of standards, standard statistics

A list of standards should be kept for each batch of standards made by the LRC. This list should include data from this

spreadsheet and any other spreadsheets using the same batch of standards. Data can be copied from old spreadsheets

and pasted (paste special: values) into cell B40 “Copy old statistics here”.

When you run a batch of samples and enter the data from the standards on the batch spreadsheet the cells D18:L39 are

automatically filled. The data in those cells can be deleted without changing any calculations in a batch.

Once all the batches have been run for a person the average values and standard deviations should help you to

determine if there are any standards in a batch that should be excluded. The coefficient of variance (COV, =StDev/Avg)

for the 30 μmol/L and 60 μmol/L standards should not exceed 4%. The COV for all other standards should not exceed

2%.

A COV of 2% or less will change most final results by less than 1.5%, where as a COV of 5% will change most final

results by upwards of 4%, (for results less than 50% BioSi). (Based on trials with data from McGlue Batch 15 and

Northern Highlands Batch 5. Results in BioSi Standards LRC Batch 2 Statistics excel spreadsheet.)

The best way to achieve the desired COV is to remove any point greater than one standard deviation if you believe it to be

incorrect. This works best when several batches are used to calculate the averages. If new standards are made they

should be tested several times to help determine average values. All values for a batch of standards should be included

even if they are from a different person’s samples.

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If an entire batch is systematically high or low that does not always mean that the batch needs to be repeated. In that

case you should check the overlap between batches to make a decision about the batch in question.

Standards may have a limited lifetime, be sure to monitor (by graph) the absorbance values for any decrease in value

over time.

B1: Batch 1 data

This spreadsheet is for sample weight data entry and spectrophotometer absorbance (abs) data entry. This spreadsheet

also calculates the final percent biogenic silica (%BioSi) for each sample.

Each spreadsheet contains two sections, one for the standard calibration curve and one for calculating the absorbance for

each sample. The orange cells are for data entry and shouldn’t be deleted (for record keeping purposes) and the yellow

cells can be deleted or edited. White cells shouldn’t be changed as they contain the formulas needed to calculate %BioSi.

This spreadsheet is not protected because it prevents the graph selection from being editable.

As you run your standards (from low abs to high abs) enter the abs into the orange cells (A5:B13) in whole numbers. If

you edit any points in the yellow cells be sure to update the standards spreadsheet. If a point needs to be disregarded

you will need to get the calibration curve from the graph and manually enter it into spreadsheet. To do this, delete the

point from the yellow abs cells (delete the concentration too) and read the equation off the chart. The a(x^2), b(x) and c

values should be manually entered in the yellow a(x^2), b (x) and c cells (C17:E18). If you delete a point for one set of

standards you will need to enter the a(x^2), b (x) and c values by hand for both sets of standards because you deleted the

concentration (which is shared between batches) as well.

As you weigh the samples record the weight (mg) in the orange weight cells (D24:D69).

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As you run the samples in the spectrophotometer record those values in the orange cells (E24:I46 and E47:I69), in whole

numbers, for the appropriate sample and time interval.

If for some reason the time interval needs to be adjusted, it should be adjusted in the corresponding orange cells

(F23:I23) and will carry over to the yellow cells (K23:N23).

Once you have run all the samples in a batch, points that do not have the desired tolerance should be edited by using the

corresponding yellow cells (K24:N45 and K46:N69). One point can be eliminated for each sample with a low R2 value.

The tolerance can be set in cell C21, it is up to the user to select an appropriate tolerance value.

The calculated Abs values should fall between 0.03 and 1.3. These values represent the lower and upper limits of the

calibration curve. It should be noted that as values approach 1.3 there could be "loss of silica to solid surfaces remaining

in the extraction tube" or "irreversible formation of unreactive silica gels or polymers that are not molybdate reactive,”

according to Mortlock and Froelich (1989). Abs values outside of the 0.03-1.3 range will be noted in the notes column (Z).

For high %BioSi the solution is to start over with less dry sediment. The range that is flagged in the notes column can be

edited in cells C22 (minimum) and D22 (maximum).

The results, %BioSi, are displayed in column X. Review the notes column, it is editable if you have any user comments. It

will carry over to the Cum. Res. spreadsheet with a note about what batch it came from.

B10 Rep.: Batch 10 repeats only data and B11 Rep.: Batch 11 repeats only data

These spreadsheets are for repeats. Section ID and depths are all manual enter. %BioSi and notes fields will not carry

over to the Cum. Res. spreadsheet. They will need to be manually entered on that spreadsheet.

Cum. Res.: Cumulative results from all batches and Cum. Res. Chart

This spreadsheet displays all of your results on one sheet. The sample ID and %BioSi are all filled in from other sheets.

The orange B10, B10 R, B11 and B11 R columns (W:Z) are all manual enter, as well as the B10/11 Notes column (AF).

The average, standard deviation, and coefficient of variance are all calculated by formula and may be edited to exclude

specific values if needed. If you exclude a value it should be noted in the notes field! The repeats column (AD) will

flag samples that have a COV and standard deviation that exceed chosen values. To choose the COV and standard

deviation, change cells E1 and G1.

The calculations

First, create a graph of absorbance v. time:

1) 2) Time

% A

bs

orb

an

ce

y = 0.000064x + 0.029437

Time

% A

bs

orb

an

ce

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These four points represent the silica digested from the clay minerals in the sediment. Second, fit a line to these points.

The equation for a line is,

where, A = absorbance (%), m = slope (silica digested from clay), t = digestion time (min), and A0 = y-intercept or the

absorbance percent at T0 (silica digested from biogenic sources).

A0 can be converted from absorbance to concentration (μmoles/L) using the standards calibration curve. To arrive at the

standards calibration curve, plot the absorption (for standards) against known concentrations, and fit a 2nd

-order

polynomial regression line to the data points,

where A0 is substituted for x to arrive at the concentration (μmoles/L) of the silica digested from biogenic sources.

The concentration, C0, is then placed in the following equation to arrive at the % biogenic silica in the sample.

[ (

) ( ) (

)]

( )

where, Conc. = C0 = concentration of silica from biogenic sources [mol/L]

Dilution factor = 5 for aliquots diluted to 5 mL and 10 for aliquots diluted to 10 mL to account for dilution from the

original concentration in the digestion bottles [unitless]

Vol. NaOH= .038 L for samples digested in 38.0 mL NaOH

SiO2 formula weight = 60.1 [g/mol]

Dry sample weight = the measured weight of each sample in grams. [g]

The units for this equation all cancel out,

[( ) ( ) (

)]

( )

and the results is a percent.

For this procedure the equation becomes,

[ (

) ( ) (

)]

( )

The spreadsheet uses a modification of this equation with C0 in μmol/L, Vol of NaOH in mL, and dry sample weight in mg,

but accounts for these minor differences in the equation with appropriate multiplication factors.

CLEAN-UP

All digestion tubes, sample vials, and caps must be cleaned. Once they are stacked in the dish washing cages they can

be washed in large groups. The lids to the cages snap on, but are not very sturdy. It is helpful to secure them with a

couple small wires/twist-ties.

PHOTOS

19

1. Shake the cage in a bucket with tap water and liquinox soap. For all rinses be sure that the water depth is over

half the height of the cage and flip several times so all sides get submerged. Watch the vials/digestion tubes to

see that they all get submerged and cleaned. Be sure to drain the liquid from the vials/tubes as best you can (it

helps to tap, rotate, and shake lightly).

2. Shake the cage in a bucket filled with tap water to rinse.

3. Shake the cage in a bucket filled with low-purity deionized water to rinse.

4. Shake the cage in a bucket filled with ~5% HCl acid wash. To make more acid wash fill the bucket with high purity

deionized water to the water fill line. Then, in a fume hood, add acid to the acid fill line (the lines correspond to a

6:1 ratio of high purity deionized water to concentrated (37%) hydrochloric acid). The acid wash must be

neutralized with soda ash before disposing into the sink, or by pouring the wash liquid into a large tub with a

couple of pieces of limestone.

5. Shake the cage in a bucket of high purity deionized water for a final rinse. This water should be changed every 5-

7 batches and if necessary, neutralized before disposal (monitor pH with pH paper). This is most easily

accomplished by pouring the wash liquid into a large tub with a couple of pieces of limestone. The reaction

proceeds slowly and may be left unattended overnight. Soda ash may be used instead but requires constant

attention.

6. Remove the vials and digestion tubes from the cage and place, upside down, in trays or test tube racks for drying.

Place the trays and racks into the fume hood to dry before use.

The volumetric flasks, storage containers, and CombiTipsfor the Eppendorf Repeater pipette may be cleaned with

several high purity deionized water rinses only.

References/Document History

Last updated by Jessica Heck, 2011. Please email [email protected] with questions, or to enquire about per sample cost

for LacCore to process samples.

DeMaster, D.J. 1979, The marine budgets of silica and 31Si, PhD Dissertation, 308 pp., Yale University, New Haven.

Mann, R. and Gieskes, J.M. 1975 Interstitial water studies, Leg 28. In: Initial Reports DSDP, 28, 805-814.

Strickland, J.D. and T.R. Parsons, 1972. Practical handbook of seawater analysis, Bull. 167, Fish. Res. Board Can. (Ottowa), 311p.

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